DNA vaccines are an innovative approach for inducing protective immunity against specific diseases encompassing the targeted delivery of plasmid DNA to cells (Montgomery, D. L. et al., 1993, Cell Biol. 169:244-247; Ulmer, J. B. et al., 1993, Science 259:1745-1749). DNA vaccines are capable of producing neutralizing antibodies, as well as inducing the more preferable cell-mediated immune (“CMI”) responses. Typically, DNA vaccines are generated by first inserting into a plasmid a gene encoding an antigen of interest, said plasmid containing a promoter active in mammalian cells. The plasmid is then transformed into a recombinant microbial host such as Escherichia coli (“E. coli”) where it is amplified and then purified. The plasmid DNA, normally suspended in saline, is administered to the body by either injection directly into muscle cells or by particle bombardment. The plasmid DNA internalized by the muscle cells is transcribed and translated, and the expressed protein is transported to the cell's surface for T-cell presentation. This mode of action results in subsequent humoral and CMI responses against the expressed antigen. Importantly, the administered plasmid DNA is non-infectious, does not replicate, and only encodes the protein of interest. Preclinical immunogenicity and efficacy of DNA vaccines in disease models have been demonstrated for a number of infectious diseases including cancer, allergy and autoimmune diseases (for review, see Gurunathan, S. et al., Ann. Rev. Immunol. 2000; 18:927-974). Clinical trials assessing the ability of DNA vaccines to generate protective immune responses against HIV, malaria, influenza, hepatitis B and cancer have been reported (for review, see Gurunathan, S. et al., 2000, Curr. Opin. Immunol. 12:442-447; Shroff, K. et al., 1999, PSTT 2:205-212; and Restifo, N. & S. Rosenberg, 1999, Curr. Opin. Oncol. 11:50-57). Recently, mixed modality vaccines have demonstrated a promising strategy whereby DNA vaccines are combined with other gene-delivery systems. Preclinical data has shown that administering plasmid DNA as a prime, followed by another gene-based vector system encoding the same antigen as a boost, results in greater immune responses than if either vector is used for both the prime and boost.
Plasmid DNA has additionally been approved for gene therapy treatment. Gene therapy encompasses the administration of a functional gene into the body, delivery of said gene to the target cell, and expression of the therapeutic product with the intent to selectively correct or modulate disease conditions. Gene therapy represents an alternative for the prevention, treatment or cure of genetic defects. Many plasmid DNA-based gene therapy clinical trials have been initiated (for review, see Mountain, A., 2000, TIBTECH 18:119-128; and Ferber, D., 2001, Science 294:1638-1642).
For use in both polynucleotide vaccination and gene therapy regimes, genes in the form of DNA plasmids can be formulated like conventional pharmaceutical products and administered directly to patients. The potential number of human users for DNA vaccines or gene therapy to combat disease, either as part of a prophylactic or therapeutic regimen, is very high, creating a large demand for plasmid DNA. DNA vaccines for veterinary diseases will likely further increase this demand. Additionally, milligram quantities of plasmid DNA may be needed for effective treatment since it has been shown that only a small number of plasmid molecules presented to a cell reach the nucleus where the gene of interest is expressed (Leitner, W. et al., 2000, Vaccine 18:765-777). Thus, the manufacture and purification of large quantities of pharmaceutical-grade DNA is crucial.
High yield plasmid DNA production processes are necessary to fully develop and exploit the advantages that both DNA vaccine and gene therapy treatment options have to offer. For these reasons, there is a continued need to increase the productivity of plasmid DNA manufacturing and purification methodologies. Many described methods for increasing plasmid DNA production for use in gene therapy or polynucleotide vaccination focus on the plasmid purification step, i.e. the downstream part of the production process; however, much less is known about how to optimize the initial fermentation step of the production process for the generation of plasmid DNA, especially for production at an industrial scale. Despite prior investigations into small scale plasmid DNA purification methodologies, it has been difficult to scale up the manufacture and purification of clinical-grade plasmid DNA (Prazeres, D. M. F. et al., 1999, TIBTECH 17:169-174). Using non-optimized laboratory conditions for the production of plasmid DNA invariably leads to very low (5-40 mg/L) volumetric yields. Increasing the productivity of plasmid DNA manufacturing processes requires the concomitant optimization of plasmid copy number (i.e., specific yield) and biomass concentration (i.e., volumetric yield). While some techniques identified for optimizing fermentation methods for recombinant protein production by E. coli on a commercial scale may be translatable to processes aimed at the over-production of plasmid, the conditions facilitating optimal protein expression will likely differ to some degree from those necessary for achieving high plasmid copy number.
PCT International Application PCT/US96/09746 (International publication number WO 96/40905) discloses a fed-batch fermentation method for generating production scale quantities of pharmaceutical grade plasmid DNA in a microorganism at high efficiencies whereby growth rate is limited to achieve optimum yield.
PCT International Application PCT/EP98/01122 (International publication number WO 98/37179) discloses the use of chemically-defined medium for the fermentative production of valuable compounds on an industrial scale, in addition to the selection of a high growth strain on said chemically-defined medium after mutagenic treatment.
U.S. Pat. Nos. 5,981,735 and 6,503,738, issued to Thatcher et al. on Nov. 9, 1999 and Jan. 7, 2003, respectively, disclose a scalable method for the production of highly purified plasmid DNA in E. coli consisting of growing plasmid-containing cells to a high biomass in exponential growth and lysing the cells by raising the pH of the culture to a value in which chromosomal DNA is denatured but plasmid DNA is reversibly renatured.
O'Kennedy, R. et al. (2000, J. Biotechnol. 76:175-183) show that culturing E. coli DH5α cells harboring the plasmid pSVβ in a semi-defined medium results in higher plasmid specific yields over the standard complex Luria Bertrani (“LB”) medium formulation, demonstrating the existence of an optimum carbon/nitrogen ratio.
The present invention discloses a highly productive, scalable and reproducible process for the production of plasmid DNA. The process combines the selection of highly productive clones of E. coli with the induction of plasmid amplification during fermentation as a result of utilizing a limited nutrient feeding regime in a chemically-defined medium. This process is useful for the production of plasmid DNA for gene therapy and genetic vaccination for a number of human and animal diseases, including HIV, hepatitis C and rabies.